This invention relates to inhibition of replication of human immunodeficiency virus (HIV).
HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). The trans-activation region (TAR) and the Rev-response element (RRE) (Rosen et al., 1988; Dayton et al., 1989; Malim et al., 1990) of HIV are found in unspliced or partially spliced HIV mRNA introns. During replication of HIV, the RRE and TAR RNAs interact with specific HIV proteins. The RRE is recognized by the HIV protein Rev (Daly et al., 1989; Zapp and Green, 1989; Cochrane et al., 1990; Heaphy et al., 1930; Malim et al., 1990) which stimulates mRNA export from the nucleus (Emerman et al., 1989; Malim et al., 1990; Malim and Cullen 1993; Fischer et al., 1994; Meyer and Malim, 1994; Bogerd et al., 1995; Stutz et al., 1995) via the formation of a Rev/RRE complex which displays a nuclear export signal that is essential for Rev-mediated export of RNA from the nucleus and also for Rev shuttling (Malim et al., 1991; Fischer et al., 1994; Meyer and Malim, 1994; Fischer et al., 1995; Stutz et al., 1995; Wen et al., 1995; Wolff et al., 1995). The Rev/RRE interaction regulates the cytoplasmic accumulation of HIV genomic and structural mRNAs and is therefore essential if the virus is to propagate.
The RRE contains a series of stem-loop structures protruding from a long central stem, known as Stem I (Dayton et al., 1989; Malim et al., 1989b; Dayton et al., 1992; Mann et al., 1994), as shown in FIG. 1 (RRE-WT)[SEQ ID NO: 1]. At the base of Stem IIb is a high-affinity Rev-binding motif which is recognized by a single Rev protein with a Kd of approximately 1nM (Bartel et al., 1991; Heaphy et al., 1991; Iwai et al., 1992; Kjems et al., 1992; Tiley et al., 1992). This high-affinity motif is a purine-rich bubble stabilized by non-Watson-Crick Gxe2x80xa2A and Gxe2x80xa2G base pairs (Heaphy et al., 1991; Bartel et al., 1991; Iwai et al., 1992; Pritchard et al., 1994). Together with a bulged-out uridine nucleotide, these non-Watson-Crick base pairs open the major groove of the mRNA duplex and permit the recognition of functional groups on the two base pairs either side of the bulged region inside the widened major groove. In addition to these base-specific contacts, phosphate contacts are made around the bubble as well as with base-paired nucleotides further away from the bubble (Iwai et al., 1992; Kjems et al., 1992; Pritchard et al., 1994).
Mutational analysis of the RRE has shown that the high-affinity interaction with a single Rev protein is necessary, but not sufficient, for Rev activity in vivo (Dayton et al., 1989; Malim et al., 1989b; Malim et al., 1990; Olsen et al., 1990; Bartel et al., 1991; Huang et al., 1991; Dayton et al., 1992; Holland et al., 1992; Mann et al., 1994). For full activity, further Rev monomers must be able to oligomerize along stem I of the RRE (Heaphy et al., 1990, 1991; Malim and Cullen, 1991; Mann et al., 1994). Truncations of Stem I that do not affect the high-affinity motif reduce Rev responses by removing additional potential binding sites for Rev monomers, with the longest truncations producing the greatest losses of activity (Mann et al., 1994). Similarly, mutations in the Rev protein that block oligomerization along the RNA stem result in an inactive protein (Malim and Cullen, 1991; Zapp et al., 1991).
It has been suggested that up to twelve Rev monomers in total can bind to each wild-type RRE (Mann et al., 1934). The high-affinity motif is not the sole Rev binding site on the RRE, however, unless a monomer is bound to the high-affinity motif, the oligomerization of Rev cannot take place. The binding of a single Rev to the high-affinity motif facilitates the binding and co-operative oligomerization of additional Rev monomers along the RRE (Iwai et al., 1992; Mann et al., 1994), with neighboring Rev monomers in contact with one another (Mann et al., 1994).
Various models have been proposed as to the mechanism by which Rev oligomerization is achieved. Kjems et al. (1991) suggested that Rev monomers bind to a variety of sequence-specific sites in the RRE. Zapp et al. (1991) argued that Rev binds to the RRE high-affinity site as a pre-existing tetramer. Malim and Cullen (1991) ascribed the oligomerization solely to protein/protein interactions between neighboring Rev monomers, and Tiley et al. (1992) reached the same conclusion. Powell et al. (1995) refined this view, believing that sequence-specific information in the RNA can exert a subtle influence on higher-order binding, but maintain that protein/protein interactions are the major determinant directing oligomerization.
Disruption of the natural Rev/RRE interaction via mutation of the natural sequences has been explored in the prior art as a potential avenue to the use of altered Rev or RRE molecules in anti-HIV therapy. Transdominant Rev mutants which retain the RRE-binding features of wild-type Rev but which are defective in certain other features have been described(e.g. Malim et al., 1989a; Malim et al., 1991; Bogerd et al., 1995).
Harada et al. (1996) relates to in vivo methods for selecting short peptides which bind Rev.
Jensen, K. B. et ,a. (1995) disclose chemically modified RNA sequences (i.e., containing 5-iodouridine) which bind Rev in vitro with higher affinity than the RRE and which are able to crosslink with Rev at a 1:1 ratio. These are postulated as potential suicide ligands for in vivo disease inhibition, however, non-specific interactions with chemically reactive bases cannot be ruled out in an in vivo situation.
WO92/05195 discloses molecules which mimic the high-affinity binding site of the native RRE in order to act as competitive inhibitors, thus sequestering free Rev protein and preventing it from interacting with those mRNAs which contain the RRE. These molecules contain a greater number of Rev binding sites than are contained in viral RRE-containing mRNAs.
One object of the invention is to provide nucleic acid molecules which inhibit HIV replication.
Another object of the invention is to provide a nucleic acid decoy which binds HIV Rev protein so as to inhibit HIV infection.
Another object of the invention is to provide a nucleic acid decoy which binds HIV Rev protein with greater co-operativity than the wild-type RRE.
The invention is based on the unexpected discovery that model RREs comprising a high-affinity binding-motif flanked by perfect duplex RNA can only bind a monomer of Rev, and that disruptions to the RNA duplex in the vicinity of the high-affinity motif are necessary to permit the binding of additional Rev monomers. It also has been discovered that each disruption seems to allow the binding of an additional Rev monomer.
Therefore, according to the present invention there is provided an isolated nucleic acid comprising two or more operatively linked binding sites for HIV Rev protein, the sites comprising at least one nucleation motif and at least one oligomerization motif, wherein the nucleic acid binds Rev protein monomers with a higher degree of co-operativity than wild-type RRE.
As used herein, the term xe2x80x9coperatively linkedxe2x80x9d means that oligomerization of a second HIV Rev protein along a nucleic acid molecule of the invention is initiated by the sequence-specific binding of a single Rev monomer at a nucleation motif. Therefore, in order for binding of a second Rev protein to occur, the binding of a first Rev protein at a high affinity site (i.e., a nucleation motif) must occur.
As used herein, the term xe2x80x9cnucleation motifxe2x80x9d refers to a nucleic acid binding site for Rev protein, wherein the Rev occupancy of the binding site is independent of the presence of any other bound Rev monomers.
The term xe2x80x9coligomerization motifxe2x80x9d refers to a nucleic acid binding site for Rev protein, wherein the Rev occupancy of the binding site requires at least one Rev monomer to have already bound to an operatively linked Rev-binding site.
The nucleation motif is recognized by Rev in a sequence-specific manner. The nucleation motif may comprise a sequence of the following generic structural formula:
xe2x80x94EG RN AYPxe2x80x94
xe2x80x94FCRxe2x80x2(Nxe2x80x2)nGRxe2x80x3Qxe2x80x94
where n=0 or 1; Y and Rxe2x80x3 are, respectively, a pyrimidine and purine; E and F are nucleotides which can form a base pair; P and Q are nucleotides which can form a base pair; R and Rxe2x80x2 is a purine; and N and Nxe2x80x2 is any nucleotide.
The nucleation motif is preferably the RNA minimal Rev binding motif present in the native RRE, shown boxed in FIG. 1 [SEQ ID NO: 1; nucleotides 103-108 and 127-133].
As used herein, the term xe2x80x9cisolated nucleic acidxe2x80x9d refers to nucleic acids which are produced by chemical synthesis or utilizing nucleotide polymerases, in vivo or ex vivo, and refers to a plurality of joined nucleotide units, or nucleotide analogue units, but does not include a nucleic acid in its natural environment. Thus nucleic acids of the invention may contain any of the five naturally-occurring bases found in DNA and RNA, and also may contain alternative bases such as inosine, or purine or pyrimidine bases not normally found in nature, or modifications to the cycloflranose portions of bases. Isolated nucleic acids of the invention may contain some altered inter-sugar linkages, as exemplified by phosphorothioate and other sulphur-containing species well-known in the art; altered sugar moieties, such as those with substitutions at their 2xe2x80x2 position. Such 2xe2x80x2 substituents might be OH, SH, F, OCH3 groups.
It is preferred that the nucleic acid is RNA. This may be prepared by direct transcription from a coding or complementary DNA sequence in vitro or in vivo, or by chemical synthesis. RNAs according to the invention are termed RNA decoys.
The oligomerization motifs preferably comprise a double-stranded nucleic acid in which there is a small region of disruption of the duplex structure. The term xe2x80x9cdisruptionxe2x80x9d refers to any feature in the nucleic acid sequence which results in a bulge in the duplex structure, and is further defined hereinbelow.
Preferably, the nucleic acid has a single nucleation motif and preferably one or more oligomerization motifs. The oligomerization motifs may be upstream or downstream of the nucleation motif, and may be the same sequence or a different sequence of nucleotides.
A nucleic acid molecule may also be provided which contains two or more nucleation motifs, which may be the same or different in sequence, some or all of which may be functionally linked to one or more oligomerization motifs.
The invention also encompasses a composition comprising an isolated nucleic acid as described above in combination with another anti-HIV agent, for example, a Tat decoy.
The invention also encompasses an assay for the oligomerization of Rev using a nucleic acid template as described herein, and also encompasses an assay for screening for a candidate inhibitor of the oligomerization interaction between Rev and a nucleic acid template.
The invention thus also encompasses a screening assay for inhibition of Rev binding to a nucleic acid substrate, comprising detecting a decrease in the amount of a complex comprising Rev and a nucleic acid according to the invention in the presence of a candidate inhibitor relative to the absence of the candidate inhibitor.
The invention also encompasses an assay for identifying a candidate inhibitor of HIV, comprising contacting a nucleic acid according to the invention with Rev protein in the presence of a candidate inhibitor under conditions such that a complex is permitted to form between the nucleic acid and Rev protein, and
determining the amount of complex formed in the presence of the candidate inhibitor, wherein a determination of a decrease in the amount of complex formed in the presence of the candidate inhibitor relative to its absence is indicative of inhibition.
The invention thus also encompasses an assay for identifying a candidate inhibitor of HIV, comprising providing a nucleic acid according to the invention, Rev protein, and a candidate inhibitor, detecting formation of a complex between the nucleic acid and Rev protein in the presence and absence of the candidate inhibitor, and comparing the detected formation of complex in the presence and absence of the candidate inhibitor, wherein a difference in the detected formation of complex in the presence and absence of the candidate inhibitor is indicative of inhibition.
The invention also encompasses nucleic acid templates that may form part of an assay, or assay kit, for use in the detection of oligomerization of Rev on a nucleic acid template.
The invention thus also encompasses a kit for identifying candidate inhibitor of Rev binding to a nucleic acid molecule, comprising a nucleic acid according to the invention and packaging means therefor.
The invention also encompasses a vector which encodes an isolated nucleic acid as described herein.
Preferably, the vector is targeted to cells susceptible to infection by HIV.
In the embodiment of the invention wherein the vector is targeted to cells susceptible to infection by HIV, the vector may be targeted using a vector delivery system which specifically targets cell-specific antigens found on susceptible cells, or which targets progenitor cells so that the delivered DNA is subsequently expressed in cells of a particular lineage susceptible to infection by the virus.
The vector also may encode one or more additional anti-HIV agents, such as antisense nucleic acids or ribozymes. In such a case, the anti-HIV agents may be part of the same species or may be a different species of HIV, such as HIV-1, HIV-2, HIV-3, etc.
Alternatively, the vectors may be suitable for producing nucleic acids according to the invention which may then be purified and subsequently administered to patients.
The invention also encompasses a method of transfecting a cell, comprising transfecting a target cell with a vector according to the invention.
The invention also encompasses a host cell transformed with a vector according to the invention.
The invention also encompasses a delivery system comprising a nucleic acid molecule of the invention and means to deliver the nucleic acid molecule to a target cell.
In another embodiment of the invention, tissue-specific control sequences and tissue-specific delivery systems may be combined to provide two levels of specificity to target the therapeutic nucleic acid molecule to the appropriate cells.
The invention also encompasses a nucleic acid molecule according to the invention for use in therapy, and administration of such nucleic acid molecules for treatment of HIV infection.
Particular therapeutic uses include the treatment of a patient infected with HIV and the prophylactic treatment of individuals at risk of HIV infection. Preferably, the nucleic acid molecules may be used in association with other therapeutic agents or pharmaceuticals.
The invention also encompasses the use of a nucleic acid molecule according to the invention in the preparation of a medicament for the treatment or prophylaxis of HIV infection.
The invention also encompasses a method of treating an HIV-infected patient, comprising administering an effective amount of a nucleic acid molecule according to the invention.
Preferably, the method also comprises administering one or more additional anti-HIV agents.
The invention also encompasses a method of protecting individuals at risk of HIV infection, comprising administering an effective prophylactic amount of a nucleic acid of the invention.
The above-described method may also comprise the administration of one or more additional prophylactic agents.
The invention also encompasses a pharmaceutical composition comprising a nucleic acid according to the invention admixed with a pharmaceutically acceptable carrier.
Preferably, the composition further comprises one or more other antiviral agents.
The invention also encompasses a process for producing a pharmaceutical composition according to the invention comprising bringing a nucleic acid molecule of the present invention into association with a pharmaceutically acceptable carrier.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.